Marker for predicting acute graft versus host disease occurrence risk and application thereof

Abstract

The invention discloses a marker for predicting the occurrence risk of an acute graft versus host disease and application of the marker. According to the present invention, the pathogenic mutant genes EIF3C, EFCAB5, CARD8, KRT24, MAGEC1 and SPATA31C1, which are related to the occurrence of the acute graft versus host disease after transplantation, in the donor and the recipient are screened through the whole genome sequencing, wherein the pathogenic mutant genes comprise the gene EIF3C, the gene EFCAB5, the gene CARD8, the gene KRT24, the gene MAGEC1 and the gene SPATA31C1; on one hand, the risk of acute graft-versus-host disease of a transplanted patient can be predicted according to whether a pathogenic mutant gene in peripheral blood of a donor or a recipient is mutated or not, and a basis is provided for potential aGVHD diagnosis. On the other hand, a new possible mechanism is provided for the attack of the acute graft versus host disease, and a possible drug target is provided for searching for treatment of aGVHD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to markers for predicting the risk of acute graft-versus-host disease and their applications, and in particular to the application of a series of genes as evaluation indicators or markers in clinical prediction of the risk of acute graft-versus-host disease in patients after allogeneic hematopoietic cell transplantation. Background Art

[0002] Allogeneic hematopoietic cell transplantation is still the only effective and even curative treatment for hematological malignancies. Acute graft-versus-host disease (aGVHD) is a common complication after transplantation that can accumulate in one or more organs, mainly including the skin, intestines, and liver. Maculopapular rash on the back of the palm is often the first sign of aGVHD. Gastrointestinal accumulation usually appears within a few weeks after the onset of the skin, and can also appear alone without skin and liver involvement. The onset is often manifested by nausea, vomiting, and green watery stools. In severe cases, it is accompanied by severe abdominal pain and bloody stools.

[0003] aGVHD is one of the main causes of transplant-related death, with an incidence of 40% to 60% and a mortality rate of nearly 15%. Studying the impact of donor and recipient genetic changes on aGVHD after transplantation will help further reveal the molecular mechanism of acute graft-versus-host disease, increase early warning indicators and potential intervention targets, and is of vital importance for improving the prognosis and quality of life of hematopoietic cell transplant recipients. Summary of the invention

[0004] The purpose of the present invention is to provide variant genes associated with the occurrence of acute graft-versus-host disease and their application in predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation and in screening donors before hematopoietic cell transplantation.

[0005] In order to achieve the above-mentioned object, the present invention first provides a new use of a substance for detecting whether a donor gene and / or a recipient gene has mutated.

[0006] The present invention provides the use of a substance for detecting whether a donor gene and / or a recipient gene is mutated in the preparation of a product for predicting or assisting in predicting the risk of acute graft-versus-host disease in a patient after hematopoietic cell transplantation.

[0007] The present invention further provides the use of a substance for detecting whether a donor gene and / or a recipient gene is mutated in predicting or assisting in predicting the risk of acute graft-versus-host disease in a patient after hematopoietic cell transplantation.

[0008] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene has mutated in the preparation of a product for diagnosing or assisting in diagnosing whether a patient has acute graft-versus-host disease after hematopoietic cell transplantation.

[0009] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene has mutated in diagnosing or assisting in diagnosing whether a patient has acute graft-versus-host disease after hematopoietic cell transplantation.

[0010] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene is mutated in the preparation of a product for screening or assisting in screening a donor before hematopoietic cell transplantation.

[0011] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene is mutated in screening or assisting in screening donors before hematopoietic cell transplantation.

[0012] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene is mutated in preparing a product for pairing a donor and a recipient before hematopoietic cell transplantation (matching a donor and a recipient before hematopoietic cell transplantation).

[0013] The present invention also provides the use of a substance for detecting whether a donor gene and / or a recipient gene has mutated in the pairing of a donor and a recipient before hematopoietic cell transplantation (matching a donor and a recipient before hematopoietic cell transplantation).

[0014] In order to achieve the above object, the present invention further provides a kit having any one of the following functions a1)-a4):

[0015] a1) To predict or assist in predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation;

[0016] a2) To diagnose or assist in the diagnosis of acute graft-versus-host disease in patients after hematopoietic cell transplantation;

[0017] a3) Screening or assisting in the screening of donors before hematopoietic cell transplantation;

[0018] a4) Matching between donors and recipients before hematopoietic cell transplantation (matching donors and recipients before hematopoietic cell transplantation);

[0019] The kit includes substances for detecting whether a mutation occurs in a donor gene and / or a recipient gene.

[0020] In any of the above applications or kits, the donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor; the recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient.

[0021] Furthermore, the donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor's peripheral blood; the recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient's peripheral blood.

[0022] In any of the above applications or kits, the substance for detecting whether the donor gene and / or the recipient gene has mutated may be a reagent and / or an instrument for detecting whether the donor gene and / or the recipient gene has mutated.

[0023] Furthermore, the reagents and / or instruments include but are not limited to reagents for extracting genomic DNA from human peripheral blood cells, and reagents and / or instruments required for detecting whether EIF3C gene, EFCAB5 gene, CARD8 gene, KRT24 gene, MAGEC1 gene and / or SPATA31C1 gene in peripheral blood are mutated using various conventional methods in the prior art (such as DNA sequencing, restriction fragment length polymorphism, single-stranded conformation polymorphism, allele-specific oligonucleotide hybridization, etc.).

[0024] Furthermore, the reagents and / or instruments required for detecting whether the EIF3C gene, EFCAB5 gene, CARD8 gene, KRT24 gene, MAGEC1 gene and / or SPATA31C1 gene in peripheral blood are mutated by DNA sequencing (such as high-throughput sequencing or first-generation sequencing, etc.) include various primers, probes and sequencers used in sequencing.

[0025] In a specific embodiment of the present invention, the DNA sequencing method may specifically include the following steps:

[0026] 1. Draw venous blood from donors and recipients before hematopoietic cell transplantation and extract genomic DNA;

[0027] 2. Use the enzyme digestion method to randomly fragment 200ng genomic DNA into an average length of 150-200bp, then repair and fill the ends and add A bases at the 3' end to form sticky ends, then connect with the adapter containing the specific barcode sequence, and then use magnetic beads to screen to remove incomplete connection products and adapter self-connection products, and finally use universal primers complementary to the adapter sequence for PCR amplification to form a sequencing library;

[0028] 3. Use The discovery pool probes were hybridized in the hybridization buffer, and then the hybridized target fragments were captured using magnetic beads and separated and purified. The captured DNA fragments were amplified by PCR and the quality of the captured library was checked (including Qubit concentration determination and 2100 Bioanalyzer fragment length determination). Finally, PE150 high-throughput sequencing was performed on the NovaSeq6000 sequencer according to Illumina's standard process.

[0029] 4. Use sequencing profiles for genotyping.

[0030] Any of the above-mentioned kits also includes a data processing device, which has a built-in module, and the module has the following functions: predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation or diagnosing whether acute graft-versus-host disease occurs in patients after hematopoietic cell transplantation or screening donors before hematopoietic cell transplantation based on whether the EIF3C gene, EFCAB5 gene, CARD8 gene and / or KRT24 gene in the donor's peripheral blood are mutated and / or whether the MAGEC1 gene and / or SPATA31C1 gene in the recipient's peripheral blood are mutated.

[0031] Specifically,

[0032] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the EIF3C gene in the donor's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose EIF3C gene in the donor's peripheral blood is mutated is higher than that in patients after hematopoietic cell transplantation whose EIF3C gene in the donor's peripheral blood is not mutated.

[0033] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the EIF3C gene in the donor's peripheral blood is mutated is that if the EIF3C gene in the donor's peripheral blood is mutated, the patient develops or is a candidate to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the EIF3C gene in the donor's peripheral blood is not mutated, the patient does not develop or is a candidate to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0034] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the EFCAB5 gene in the donor's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose EFCAB5 gene in the donor's peripheral blood is mutated is lower than that in patients after hematopoietic cell transplantation whose EFCAB5 gene in the donor's peripheral blood is not mutated.

[0035] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the EFCAB5 gene in the donor's peripheral blood is mutated is as follows: if the EFCAB5 gene in the donor's peripheral blood is mutated, the patient does not develop or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the EFCAB5 gene in the donor's peripheral blood is not mutated, the patient develops or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0036] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the CARD8 gene in the donor's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose CARD8 gene in the donor's peripheral blood is mutated is lower than that in patients after hematopoietic cell transplantation whose CARD8 gene in the donor's peripheral blood is not mutated.

[0037] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the CARD8 gene in the donor's peripheral blood is mutated is as follows: if the CARD8 gene in the donor's peripheral blood is mutated, the patient does not develop or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the CARD8 gene in the donor's peripheral blood is not mutated, the patient develops or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0038] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the KRT24 gene in the donor's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose KRT24 gene in the donor's peripheral blood is mutated is higher than that in patients after hematopoietic cell transplantation whose KRT24 gene in the donor's peripheral blood is not mutated.

[0039] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the KRT24 gene in the donor's peripheral blood is mutated is that if the KRT24 gene in the donor's peripheral blood is mutated, the patient develops or is a candidate to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the KRT24 gene in the donor's peripheral blood is not mutated, the patient does not develop or is a candidate to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0040] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the MAGEC1 gene in the recipient's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose MAGEC1 gene in the recipient's peripheral blood is mutated is lower than that in patients after hematopoietic cell transplantation whose MAGEC1 gene in the recipient's peripheral blood is not mutated.

[0041] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the MAGEC1 gene in the recipient's peripheral blood is mutated is as follows: if the MAGEC1 gene in the recipient's peripheral blood is mutated, the patient does not develop or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the MAGEC1 gene in the recipient's peripheral blood is not mutated, the patient develops or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0042] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on whether the SPATA31C1 gene in the recipient's peripheral blood is mutated is that the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation with a mutation in the SPATA31C1 gene in the recipient's peripheral blood is lower than that in patients after hematopoietic cell transplantation without a mutation in the SPATA31C1 gene in the recipient's peripheral blood.

[0043] The method for diagnosing whether a patient develops acute graft-versus-host disease after hematopoietic cell transplantation based on whether the SPATA31C1 gene in the recipient's peripheral blood is mutated is that if the SPATA31C1 gene in the recipient's peripheral blood is mutated, the patient does not develop or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation; if the SPATA31C1 gene in the recipient's peripheral blood is not mutated, the patient develops or is likely to develop acute graft-versus-host disease after hematopoietic cell transplantation.

[0044] In practical applications, any one, any two, any three, any four, or any five of the six genes can be selected for prediction or diagnosis, or all six genes can be selected for prediction or diagnosis. When multiple genes are used for prediction or diagnosis, each gene must meet its own judgment criteria for prediction or diagnosis, otherwise the prediction or diagnosis method is invalid. For example, when the EIF3C gene and the EFCAB5 gene are used for prediction or diagnosis, if the EIF3C gene in the donor's peripheral blood is mutated and the EFCAB5 gene is not mutated, the patient will develop or be a candidate for acute graft-versus-host disease after hematopoietic cell transplantation. If the EIF3C gene in the donor's peripheral blood is mutated and the EFCAB5 gene is mutated, the prediction method is invalid.

[0045] When all six genes are used for prediction or diagnosis, in addition to prediction or diagnosis by detecting the mutations of the six genes in the donor and the recipient, prediction or diagnosis can also be performed according to the following methods:

[0046] First, the total number of gene mutations is obtained based on the mutation status of EIF3C gene, EFCAB5 gene, CARD8 gene and KRT24 gene in the donor's peripheral blood and the mutation status of MAGEC1 gene and SPATA31C1 gene in the recipient's peripheral blood. Then, the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation or whether acute graft-versus-host disease will occur in patients after hematopoietic cell transplantation is predicted based on the total number of gene mutations and the threshold.

[0047] The method for obtaining the total number of gene mutations is as follows: if the EIF3C gene, EFCAB5 gene, CARD8 gene or KRT24 gene in the donor's peripheral blood is mutated or the MAGEC1 gene or SPATA31C1 gene in the recipient's peripheral blood is mutated, it is recorded as 1, and the mutation scores of the six genes are accumulated to obtain the total number of gene mutations.

[0048] The method for predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation based on the total number of gene mutations and the threshold is as follows: the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation whose total number of gene mutations is greater than or equal to the threshold is higher than that in patients after hematopoietic cell transplantation whose total number of gene mutations is less than the threshold.

[0049] The method for diagnosing whether acute graft-versus-host disease occurs in a patient after hematopoietic cell transplantation based on the total number of gene mutations and a threshold is as follows: if the total number of gene mutations is greater than or equal to the threshold, the patient has occurred or is a candidate for occurring acute graft-versus-host disease after hematopoietic cell transplantation; if the total number of gene mutations is less than the threshold, the patient has not occurred or is a candidate for not occurring acute graft-versus-host disease after hematopoietic cell transplantation.

[0050] The threshold may specifically be 1.5.

[0051] In practical applications, the risk of acute graft-versus-host disease after hematopoietic cell transplantation can be first predicted according to the above method, and then the donors before hematopoietic cell transplantation can be screened according to the risk of acute graft-versus-host disease, or other clinical indicators can be combined to assist in the screening of donors before hematopoietic cell transplantation, so as to optimize the selection of donors before hematopoietic cell transplantation and improve the survival rate and quality of life of patients before hematopoietic cell transplantation.

[0052] Mutations in any of the above genes include any base mutations that can change the amino acid sequence of the protein encoded by the gene. The base mutations include base insertions, base deletions, base substitutions, etc. The number of base mutations includes 1 or 2 or more base mutations.

[0053] Specifically,

[0054] The EIF3C gene mutation includes at least one of the following mutations: gene fragment deletion caused by mutation at chr16-28727173; gene fragment deletion caused by mutation at chr16-28388130; gene missense mutation caused by mutation at chr16-28723250; gene frameshift insertion caused by mutation at chr16-28392031; gene frameshift insertion caused by mutation at chr16-28392043; Gene fragment deletion caused by mutation; gene missense mutation caused by mutation at chr16-28392046 site; gene missense mutation caused by mutation at chr16-28392056 site; gene missense mutation caused by mutation at chr16-28392020 site; gene missense mutation caused by mutation at chr16-28723313 site; gene frameshift insertion caused by mutation at chr16-28392043 site; gene frameshift deletion caused by mutation at chr16-28392025 site.

[0055] The EFCAB5 gene mutation includes at least one of the following mutations: a gene missense mutation caused by a mutation at the chr17-29993230 site; a gene missense mutation caused by a mutation at the chr17-29969309 site; a gene fragment deletion caused by a mutation at the chr17-29993284 site; a gene fragment deletion caused by a mutation at the chr17-29969251 site; a gene missense mutation caused by a mutation at the chr17-29993230 site; a gene missense mutation caused by a mutation at the chr17-29969309 site; a gene fragment deletion caused by a mutation at the chr17-30078487 site Missense mutation caused by point mutation; missense mutation caused by chr17-30051184 site mutation; missense mutation caused by chr17-30051119 site mutation; missense mutation caused by chr17-30057709 site mutation; missense mutation caused by chr17-30090638 site mutation; missense mutation caused by chr17-30092059 site mutation; missense mutation caused by chr17-30090516 site mutation; missense mutation caused by chr17-30078356 site mutation.

[0056] The CARD8 gene mutation includes at least one of the following mutations: gene fragment deletion caused by mutation at chr19-48234449; gene missense mutation caused by mutation at chr19-48230957; gene missense mutation caused by mutation at chr19-48234507; gene frameshift deletion caused by mutation at chr19-48231760.

[0057] The KRT24 gene mutation includes at least one of the following mutations: a gene missense mutation caused by a mutation at the chr17-40699466 site; a gene missense mutation caused by a mutation at the chr17-40699520 site; a gene missense mutation caused by a mutation at the chr17-40701194 site; a gene missense mutation caused by a mutation at the chr17-40703444 site; and a gene frameshift deletion caused by a mutation at the chr17-40701882 site.

[0058] The MAGEC1 gene mutation includes at least one of the following mutations: a missense mutation caused by a mutation at the chrX-141906385 site; a missense mutation caused by a mutation at the chrX-141906494 site; a missense mutation caused by a mutation at the chrX-141905856 site; a missense mutation caused by a mutation at the chrX-141905478 site; a missense mutation caused by a mutation at the chrX-141907529 site; a missense mutation caused by a mutation at the chrX-141906126 site; a missense mutation caused by a mutation at the chrX-141906066 site; a missense mutation caused by a mutation at the chrX-141905959 site; a missense mutation caused by a mutation at the chrX-141905478 site; and a frameshift insertion caused by a mutation at the chrX-141906056 site.

[0059] The SPATA31C1 gene mutation includes at least one of the following mutations: gene fragment deletion caused by chr9-87923373 site mutation; gene missense mutation caused by chr9-87919291 site mutation; gene missense mutation caused by chr9-87919297 site mutation; gene missense mutation caused by chr9-87922513 site mutation; gene missense mutation caused by chr9-87922941 site mutation; gene missense mutation caused by chr9-87922977 site mutation; gene frameshift insertion caused by chr9-87919289 site mutation; gene frameshift insertion caused by chr9-87919291 site mutation; gene frameshift deletion caused by chr9-87919275 site mutation; gene frameshift deletion caused by chr9-87919290 site mutation.

[0060] The fact that any of the above genes have not undergone mutations includes the fact that there are no base mutations (completely identical to the wild-type sequence of the gene) or any base mutations that do not change the amino acid sequence of the protein encoded by the gene.

[0061] In any of the above applications or kits, the donor is a healthy individual who provides hematopoietic cells before hematopoietic cell transplantation, and the recipient is a patient before hematopoietic cell transplantation.

[0062] In any of the above uses or kits, the hematopoietic cell transplantation is allogeneic hematopoietic cell transplantation.

[0063] The allogeneic hematopoietic cell transplantation is HLA haplotype-matched allogeneic hematopoietic cell transplantation.

[0064] The patient before hematopoietic cell transplantation may be an acute myeloid leukemia patient, an acute lymphoid leukemia patient, a chronic myeloid leukemia patient, a chronic lymphoid leukemia patient, a multiple myeloma patient, a myelodysplastic syndrome patient or a primary systemic amyloidosis patient.

[0065] The use of EIF3C gene and / or EFCAB5 gene and / or CARD8 gene and / or KRT24 gene and / or MAGEC1 gene and / or SPATA31C1 gene as targets in the preparation of products for preventing and / or treating acute graft-versus-host disease also falls within the protection scope of the present invention.

[0066] The ID of any of the above EIF3C genes in NCBI is 8663.

[0067] The ID of any of the above EFCAB5 genes in NCBI is 37476.

[0068] The ID of any of the above CARD8 genes in NCBI is 22900.

[0069] The ID of any of the above KRT24 genes in NCBI is 192666.

[0070] The ID of any of the above MAGEC1 genes in NCBI is 9947.

[0071] The ID of any of the above SPATA31C1 genes in NCBI is 441452.

[0072] The present invention screened out pathogenic mutation genes EIF3C, EFCAB5, CARD8, KRT24, MAGEC1, and SPATA31C1 related to the occurrence of aGVHD after transplantation through whole genome sequencing. On the one hand, the risk of acute graft-versus-host disease in patients after transplantation can be predicted based on whether the pathogenic mutation genes in the peripheral blood of donors or recipients are mutated, providing a basis for potential diagnosis of aGVHD. On the other hand, the present invention provides a new possible mechanism for the onset of aGVHD and provides a possible drug target for the search for treatment of aGVHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1The distribution of the total number of mutations in the EIF3C gene, EFCAB5 gene, CARD8 gene and KRT24 gene in the donor's peripheral blood and the MAGEC1 gene and SPATA31C1 gene in the recipient's peripheral blood in the aGVHD-positive group and the aGVHD-negative group.

[0074] Figure 2 This is the ROC curve diagram for the diagnosis of aGVHD based on the total number of mutations in the EIF3C gene, EFCAB5 gene, CARD8 gene and KRT24 gene in the donor's peripheral blood and the MAGEC1 gene and SPATA31C1 gene in the recipient's peripheral blood. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0076] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0077] The HLA haplotype matching involved in the present invention means that the HLA-A, HLA-B, and HLA-DRB1 alleles between the related donor and recipient are the same on the same chromosome, but at least one of the above alleles on another chromosome is different (i.e. 3 / 6, 4 / 6, or 5 / 6 matching).

[0078] Example 1. Screening of donor and recipient mutant genes associated with aGVHD

[0079] 1. Screening of donor and recipient mutation genes associated with aGVHD

[0080] 1. Research subjects

[0081] The research subjects of this example are as follows: peripheral blood samples of 65 patients with aGVHD and their donors (defined as the aGVHD positive group), and peripheral blood samples of 65 patients without aGVHD and their donors (defined as the aGVHD negative group).

[0082] Inclusion criteria: 1) Patients underwent HLA haplotype-matched allogeneic hematopoietic cell transplantation at Peking University People's Hospital in 2019; 2) Patients had malignant blood diseases before transplantation, including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, myelodysplastic syndrome, and primary systemic amyloidosis; 3) Patients and donors were aged between 14 and 65 years old; 4) The aGVHD-positive group included patients diagnosed with aGVHD 2-4 within 100 days after transplantation; the aGVHD-negative group included patients who did not develop aGVHD symptoms within 100 days after transplantation and were paired 1:1 with the aGVHD-positive group. There was no statistical difference in age and gender between the two groups.

[0083] Exclusion criteria: 1) The patient's disease before transplantation was not acute promyelocytic leukemia or aplastic anemia; 2) This was not the first hematopoietic cell transplantation for the patient; 3) The patient died within three months after transplantation and the cause of death was not diagnosed as aGVHD.

[0084] 2. Sample preparation

[0085] The genomic DNA was extracted from the collected peripheral blood samples using the phenol-chloroform method to test the purity, concentration, integrity and contamination of the DNA. The qualified samples were sequenced for genome sequencing, and the experimental process was carried out according to the library preparation steps and sequencing steps provided by Illumina.

[0086] 3. Next-generation sequencing

[0087] The library was constructed using the human whole exome sequence capture kit Truseq Nano, and hybridized with probes for capture and enrichment. The samples were then sequenced using the Illumina Novaseq S4 PE150 high-throughput sequencing platform, with an average sequencing depth of 35x for each sample.

[0088] 4. Results Analysis

[0089] FastQC software was used to assess the quality of the raw data, and BWA (parameter-M) software was used for genome alignment. All analyses were performed based on the human reference genome hg38.

[0090] 2. Acquisition of donor and recipient mutant genes associated with aGVHD

[0091] In order to analyze the correlation between mutant genes and the occurrence of aGVHD, the relationship between mutations that change protein functional structure and aGVHD was systematically analyzed. In this study, mutations that affect protein structure annotated by ANNOVAR were defined as protein loss of function mutations (LOF mutations). The ANNOVAR annotations included "stopgain (premature termination of gene coding)", "startloss (start codon deletion)", "frameshift insertion (frameshift insertion)", "frameshift deletion (frameshift deletion)" and "frameshift block substitution (frameshift mutation)". Finally, 4 donor LoF mutant genes (EIF3C, EFCAB5, CARD8, KRT24) and 2 recipient LoF mutant genes (MAGEC1, SPATA31C1) were identified in the whole genome as being associated with aGVHD (p<0.005). The relevant information of each gene is shown in Table 1.

[0092] Table 1

[0093] Gene Gene ID Gene name Location EIF3C 8663 eukaryotic translation initiation factor 3subunit C Chromosome 16 EFCAB5 374786 EF-hand calcium binding domain 5 Chromosome 17 CARD8 22900 caspase recruitment domain family member 8 Chromosome 19 KRT24 192666 keratin 24 Chromosome 17 MAGEC1 9947 MAGE family member C1 Chromosome X SPATA31C1 441452 SPATA31 subfamily C member 1 Chromosome 9

[0094] 3. Analysis of mutations of the six genes in samples of the aGVHD-positive or aGVHD-negative groups

[0095] The mutation status of the 6 genes in the collected samples is as follows:

[0096] 1. Among the 130 donors, 13 had EIF3C gene mutations, including 10 in the aGVHD-positive group and 3 in the aGVHD-negative group. The aGVHD groups and the EIF3C gene status of the donors are shown in Table 2. After chi-square test analysis, P=0.041, there was a statistically significant difference between the two groups.

[0097] Table 2

[0098]

[0099] The specific mutations of EIF3C gene in 13 donors included mutations at chr16-28727173 and chr16-28388130 causing gene fragment deletion; mutations at chr16-28723250 causing gene missense mutation; and mutations at chr16-28392031 and chr16-28392043 causing gene frameshift insertion.

[0100] 2. Among the 130 donors, 7 had EFCAB5 gene mutations, all of whom were in the aGVHD negative group. The aGVHD group and the EFCAB5 gene status of the donors are shown in Table 3. After chi-square test analysis, P=0.007, there was a statistically significant difference between the two groups.

[0101] Table 3

[0102]

[0103] The specific mutations of the EFCAB5 gene in the 7 donors included mutations at chr17-29993230 and chr17-29969309, which resulted in missense mutations.

[0104] 3. Among the 130 donors, 31 had CARD8 gene mutations, including 10 donors in the aGVHD-positive group and 21 donors in the aGVHD-negative group. The aGVHD group and the CARD8 gene status of the donors are shown in Table 4. After chi-square test analysis, P=0.024, there was a statistically significant difference between the two groups.

[0105] Table 4

[0106]

[0107] The specific mutations of the CARD8 gene in the 31 donors included mutations at chr19-48234449 resulting in gene fragment deletion; mutations at chr19-48230957 and chr19-48234507 resulting in gene missense mutations; and mutations at chr19-48231760 resulting in gene frameshift deletion.

[0108] 4. A total of 50 of the 130 donors had KRT24 gene mutations, including 33 donors in the aGVHD-positive group and 17 donors in the aGVHD-negative group. The aGVHD grouping and the KRT24 gene status of the donors are shown in Table 5. After chi-square test analysis, P=0.004, there was a statistically significant difference between the two groups.

[0109] Table 5

[0110]

[0111]

[0112] The specific mutations of KRT24 gene in 50 donors included mutations at chr17-40699466, chr17-40699520, chr17-40701194 and chr17-40703444, which resulted in missense mutations; mutation at chr17-40701882 resulted in frameshift deletion.

[0113] 5. Among the 130 recipients, 4 cases had MAGEC1 gene mutations, all of whom were recipients in the aGVHD negative group; the aGVHD group and the MAGEC1 gene status of the recipients are shown in Table 6. After chi-square test analysis, P=0.042, there was a statistically significant difference between the two groups.

[0114] Table 6

[0115]

[0116] The specific mutations of the MAGEC1 gene in the four recipients included mutations at chrX-141906385, chrX-141906494, chrX-141905856 and chrX-141905478, which resulted in missense mutations; the mutation at chrX-141906056 resulted in frameshift insertion.

[0117] 6. Among the 130 recipients, 10 had SPATA31C1 gene mutations, all of whom were in the aGVHD negative group. The aGVHD group and the SPATA31C1 gene status of the recipients are shown in Table 7. After chi-square test analysis, P=0.001, there was a statistically significant difference between the two groups.

[0118] Table 7

[0119]

[0120] The somatic mutations of the SPATA31C1 gene in the 10 recipients included mutations at chr9-87923373 resulting in gene fragment deletion; mutations at chr9-87919291, chr9-87922941 and chr9-87922977 resulting in gene missense mutations; mutations at chr9-87919289 and chr9-87919291 resulting in gene frameshift insertions; and mutations at chr9-87919275 and chr9-87919290 resulting in gene frameshift deletions.

[0121] Example 2: Verification of donor and recipient mutant genes associated with aGVHD

[0122] 1. Screening of donor and recipient mutation genes associated with aGVHD

[0123] 1. Research subjects

[0124] The research subjects of this example are as follows: peripheral blood samples of 106 patients with aGVHD and their donors (defined as the aGVHD positive group), and peripheral blood samples of 106 patients without aGVHD and their donors (defined as the aGVHD negative group).

[0125] Inclusion criteria: 1) Patients underwent HLA haplotype-matched allogeneic hematopoietic cell transplantation at Peking University People's Hospital from 2018 to 2019; 2) Patients had malignant blood diseases before transplantation, including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, myelodysplastic syndrome, and primary systemic amyloidosis; 3) Patients and donors were aged between 14 and 65 years old; 4) The aGVHD-positive group included patients diagnosed with aGVHD 2-4 within 100 days after transplantation; the aGVHD-negative group included patients who did not develop aGVHD symptoms within 100 days after transplantation and were paired 1:1 with the aGVHD-positive group. There was no statistical difference in age and gender between the two groups.

[0126] Exclusion criteria: 1) The patient's disease before transplantation was not acute promyelocytic leukemia or aplastic anemia; 2) This was not the first hematopoietic cell transplantation for the patient; 3) The patient died within three months after transplantation and the cause of death was not diagnosed as aGVHD.

[0127] 2. Sample preparation

[0128] The genomic DNA was extracted from the collected peripheral blood samples using the phenol-chloroform method to test the purity, concentration, integrity and contamination of the DNA. The qualified samples were sequenced for genome sequencing, and the experimental process was carried out according to the library preparation steps and sequencing steps provided by Illumina.

[0129] 3. Next-generation sequencing

[0130] The library was constructed using the human whole exome sequence capture kit Truseq Nano, and hybridized with probes for capture and enrichment. The samples were then sequenced using the Illumina Novaseq S4 PE150 high-throughput sequencing platform, with an average sequencing depth of 35x for each sample.

[0131] 4. Analysis

[0132] FastQC software was used to assess the quality of the raw data, and BWA (parameter-M) software was used for genome alignment. All analyses were performed based on the human reference genome hg38.

[0133] II. Analysis of mutations of the six genes in samples of the aGVHD positive or negative groups

[0134] The mutation status of the 6 genes in the collected samples is as follows:

[0135] 1. A total of 15 of the 212 donors had EIF3C gene mutations, including 12 donors in the aGVHD-positive group and 3 donors in the aGVHD-negative group. The aGVHD grouping and the EIF3C gene status of the donors are shown in Table 8. After chi-square test analysis, P=0.016, there was a statistically significant difference between the two groups.

[0136] Table 8

[0137]

[0138] The specific mutations of EIF3C gene in 15 donors included mutations at chr16-28727173, chr16-28388130, chr16-28727173, chr16-28388130 and chr16-28391825, which resulted in gene fragment deletion; mutations at chr16-28723250, chr16-28392046, chr16-28392056, chr16-28392020 and chr16-28723313, which resulted in gene missense mutation; mutations at chr16-28392031 and chr16-28392043, which resulted in gene frameshift insertion; and mutation at chr16-28392025, which resulted in gene frameshift deletion.

[0139] 2. A total of 12 of the 212 donors had EFCAB5 gene mutations, including 1 donor in the aGVHD-positive group and 11 donors in the aGVHD-negative group. The aGVHD grouping and the EFCAB5 gene status of the donors are shown in Table 9. After chi-square test analysis, P=0.003, there was a statistically significant difference between the two groups.

[0140] Table 9

[0141]

[0142] The specific mutations of the EFCAB5 gene in the 12 donors included mutations at chr17-29993284 and chr17-29969251, which resulted in gene fragment deletion; mutations at chr17-29993230, chr17-29969309, chr17-30078487, chr17-30051184, chr17-30051119, chr17-30057709, chr17-30090638, chr17-30092059, chr17-30090516 and chr17-30078356, which resulted in gene missense mutations.

[0143] 3. A total of 49 of the 212 donors had CARD8 gene mutations, including 14 donors in the aGVHD-positive group and 35 donors in the aGVHD-negative group. The aGVHD grouping and the CARD8 gene status of the donors are shown in Table 10. After chi-square test analysis, P=0.001, there was a statistically significant difference between the two groups.

[0144] Table 10

[0145]

[0146]

[0147] The specific mutations of the CARD8 gene in the 49 donors included mutations at chr19-48234449 resulting in gene fragment deletion; mutations at chr19-48230957 and chr19-48234507 resulting in gene missense mutations; and mutations at chr19-48231760 resulting in gene frameshift deletion.

[0148] 4. A total of 81 of the 212 donors had KRT24 gene mutations, including 52 donors in the aGVHD-positive group and 29 donors in the aGVHD-negative group. The aGVHD grouping and the KRT24 gene status of the donors are shown in Table 11. After chi-square test analysis, P=0.001, there was a statistically significant difference between the two groups.

[0149] Table 11

[0150]

[0151] The specific mutations of KRT24 gene in 81 donors included mutations at chr17-40699466, chr17-40699520, chr17-40701194 and chr17-40703444, which resulted in missense mutations; mutation at chr17-40701882 resulted in frameshift deletion.

[0152] 5. Among the 212 recipients, 8 cases had MAGEC1 gene mutations, all of whom were recipients in the aGVHD negative group; the aGVHD group and the MAGEC1 gene status of the recipients are shown in Table 12. After chi-square test analysis, P=0.042, there was a statistically significant difference between the two groups.

[0153] Table 12

[0154]

[0155] The specific mutations of the MAGEC1 gene in the eight recipients included mutations at chrX-141906385, chrX-141907529, chrX-141906126, chrX-141906066, chrX-141905959 and chrX-141905478, resulting in missense mutations of the gene.

[0156] 6. Among the 212 recipients, 19 had SPATA31C1 gene mutations, including 3 recipients in the aGVHD-positive group and 16 recipients in the aGVHD-negative group. The aGVHD grouping and the SPATA31C1 gene status of the recipients are shown in Table 13. After chi-square test analysis, P=0.002, indicating a statistically significant difference between the two groups.

[0157] Table 13

[0158]

[0159] The somatic mutations of the SPATA31C1 gene in the 19 recipients included mutations at chr9-87923373 resulting in gene fragment deletion; mutations at chr9-87919297, chr9-87922513, chr9-87922941 and chr9-87922977 resulting in gene missense mutations; mutations at chr9-87919289 and chr9-87919291 resulting in gene frameshift insertions; and mutations at chr9-87919275 and chr9-87919290 resulting in gene frameshift deletions.

[0160] Example 3: Prediction of aGVHD by combination of donor and recipient mutant genes

[0161] The 212 patients and their donor peripheral blood samples in Example 2 were used as research objects, and the total number of gene mutations for each patient was obtained according to the following method: if the EIF3C, EFCAB5, CARD8 or KRT24 gene in the donor peripheral blood was mutated or the MAGEC1 or SPATA31C1 gene in the recipient peripheral blood was mutated, it was recorded as 1, and the mutation scores of the six genes were accumulated, that is, the total number of gene mutations; if the EIF3C, EFCAB5, CARD8 and KRT24 genes in the donor peripheral blood were not mutated and the MAGEC1 or SPATA31C1 genes in the recipient peripheral blood were not mutated, it was recorded as 0, that is, the total number of gene mutations was 0.

[0162] The Mann-Whitney U test was performed to compare the total number of gene mutations and the occurrence of aGVHD in the 212 patients. The statistical difference between the two groups of aGVHD was compared, and the receiver operating characteristic (ROC) curve was used to evaluate the specificity and sensitivity of diagnosis. The Yoden Index was used to calculate the cut-off value of diagnosis.

[0163] After analysis, it was found that the total number of gene mutations in the aGVHD-positive group was significantly higher than that in the aGVHD-negative group ( Figure 1 , P<0.001). ROC curve analysis showed that the area under the curve of the total number of gene mutations for diagnosing aGVHD was 0.743 ( Figure 2 , 95% confidence interval was 0.677-0.808), the total number of gene mutations corresponding to the maximum Youden index was 1.5. Taking this as the diagnostic threshold of aGVHD, the overexpression rate of the total number of gene mutations in aGVHD was 65%.

[0164] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. The use of substances for detecting whether donor genes and / or recipient genes are mutated in the preparation of products for predicting or assisting in predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation; Or, the use of a substance for detecting whether a donor gene and / or a recipient gene has been mutated in predicting or assisting in predicting the risk of acute graft-versus-host disease in a patient after hematopoietic cell transplantation; The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient.

2. The use of substances for detecting whether the donor gene and / or recipient gene has mutated in the preparation of products for diagnosing or assisting in diagnosing whether a patient has acute graft-versus-host disease after hematopoietic cell transplantation; Or, the use of a substance for detecting whether a donor gene and / or a recipient gene has mutated in diagnosing or assisting in diagnosing whether a patient has developed acute graft-versus-host disease after hematopoietic cell transplantation; The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient.

3. The use of substances for detecting whether the donor gene and / or recipient gene has mutated in the preparation of products for screening or assisting in screening donors before hematopoietic cell transplantation; Or, the use of a substance for detecting whether a donor gene and / or a recipient gene has been mutated in screening or assisting in screening donors before hematopoietic cell transplantation; Or, use of a substance for detecting whether a donor gene and / or a recipient gene has been mutated in preparing a product for matching a donor and a recipient before hematopoietic cell transplantation; or, the use of a substance for detecting whether a donor gene and / or a recipient gene has been mutated in matching a donor and a recipient before hematopoietic cell transplantation; The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient.

4. The use according to any one of claims 1 to 3, characterized in that: The hematopoietic cell transplantation is allogeneic hematopoietic cell transplantation.

5. The use according to any one of claims 1 to 4, characterized in that: The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor's peripheral blood; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient's peripheral blood.

6. A kit having any one of the functions a1) to a4) below, comprising a substance for detecting whether a donor gene and / or a recipient gene has mutated; a1) To predict or assist in predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation; a2) To diagnose or assist in the diagnosis of acute graft-versus-host disease in patients after hematopoietic cell transplantation; a3) Screening or assisting in the screening of donors before hematopoietic cell transplantation; a4) Matching between donor and recipient before hematopoietic cell transplantation; The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient.

7. The kit according to claim 6, characterized in that: The hematopoietic cell transplantation is allogeneic hematopoietic cell transplantation.

8. The kit according to claim 6 or 7, characterized in that: The donor gene is the EIF3C gene and / or the EFCAB5 gene and / or the CARD8 gene and / or the KRT24 gene in the donor's peripheral blood; The recipient gene is the MAGEC1 gene and / or the SPATA31C1 gene in the recipient's peripheral blood.

9. The kit according to any one of claims 6 to 8, characterized in that: The kit also includes a data processing device, which has a module inside. The module has the following functions: predicting the risk of acute graft-versus-host disease in patients after hematopoietic cell transplantation or diagnosing whether acute graft-versus-host disease occurs in patients after hematopoietic cell transplantation or screening donors before hematopoietic cell transplantation based on whether EIF3C gene and / or EFCAB5 gene and / or CARD8 gene and / or KRT24 gene in the donor's peripheral blood are mutated and / or whether MAGEC1 gene and / or SPATA31C1 gene in the recipient's peripheral blood are mutated.

10. Use of EIF3C gene and / or EFCAB5 gene and / or CARD8 gene and / or KRT24 gene and / or MAGEC1 gene and / or SPATA31C1 gene as targets in the preparation of products for preventing and / or treating acute graft-versus-host disease.

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